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First Annual IFT Food Protection & Defense Research Conference Atlanta Marriot Marquis November 2-4, 2005 Thomas A. Cebula, Ph.D. Director, Office of Applied Research and Safety Assessment CFSAN The TIGER Biosensor: Rapid Broad Range Pathogen Detection in Diagnostics and Food Protection Lawrence Blyn, Ph.D., Ibis Therapeutics Improved diagnostic tests for avian influenza surveillance Blanca Lupiani, Ph.D., Texas A&M University Efficient nucleic signature development for broad spectrum pathogen detection Jason Gans, Ph.D., Los Alamos National Lab Expanding the Use of Validated Rapid Microbiological Methods to New Food Matrices Willis Fedio, Ph.D., New Mexico State University “Before beginning a Hunt, it is wise to ask someone what you are looking for before you begin looking for it.” --A.A. Milne, 1926, Pooh's Little Instruction Book The need for: •Identifying and recognizing patterns in a disease outbreak •Communicating those patterns to the public health community at large •Determining the pathogen involved •Containing the outbreak •Tracing the microbe to its source --Events of 9/11/2001 and after The forensic continuum for strain identification “Strain could not have come from…” “Strain did absolutely come from…” Exclusion Attribution Methods Validation Differentiation of Strains Biomarker Stability Extent of Genomic Diversity Food Safety Detection at Genus Level Detection at Species Level Detection at Subspecies Level Detection at Serotype or Serovar Level Food Defense Detection at Genus Level Detection at Species Level Detection at Subspecies Level Detection at Serotype or Serovar Level but, for attribution, Detection at Strain Level Bacterial Diversity Whose strains define the universe of diversity that we study? There is a genuine lack of appreciation concerning the extent of diversity that exists among plant and animal pathogens. At this writing, with the complete sequence of four bacterial genomes already known and a fifth, that of E. We examine basic tenets of Cebula LeClerc, coli, to be unveiled shortly, some still myopically evolution, i.e., the relative roles 1997 question whether bacteria genomics will offer many that mutation and recombination surprises. The Salmonella sequencing project has been play in instituting the genetic impacted, hampered by the belief that Salmonella is too diversity upon which selection much like E. coli to warrant intense effort. This apathy works to establish bacteria in seems steeped in the naive assumption that experiments particular niches. Specifically, we conducted in an unnatural setting (the test tube) can be delve into the importance of correlated directly with how bacteria behave in their particular mutator phenotypes and natural environment. That we do not share this belief is their potential contributions obvious—to do so belies an appreciation of their to homeologous recombination in differences. bacteria. The implications for Homeologous Recombination: Ingredients for Rapid Evolution rapid Hypermutability evolution& and the emergence of new pathogens are discussed. Cebula and LeClerc, 1997 265 Microbial Genomes Sequenced • 21 Archaea • 211 Bacteria • 33 Eukaryotes In Progress • 1470 Microbial Genomes www.Genomesonline.org As of June 2, 2005 298 Microbial Genomes Sequenced • 23 Archaea • 236 Bacteria • 39 Eukaryotes In Progress • 1589 Microbial Genomes www.Genomesonline.org As of September 16, 2005 303 Microbial Genomes Sequenced • 24 Archaea • 240 Bacteria • 39 Eukaryotes In Progress • 1608 Microbial Genomes www.Genomesonline.org As of October 20, 2005 313 Microbial Genomes Sequenced • 25 Archaea • 249 Bacteria • 39 Eukaryotes In Progress • 1686 Microbial Genomes www.Genomesonline.org As of October 25, 2005 100 100 Prior Agreement 3 Housekeeping 100 100 100 90 100 96 100 100 100 100 74 100 73 99 100 100 95 77 100 100 100 69 100 100 100 100 S3044 S3041 S2979 S2978 S3014 S3013 S3027 S3015 S3057 S2995 S4194 S3333 S2985 S2993 S2983 S2980 EC52 EC64 V S. bongori IIIb VII IV S. enterica I VI S. typhimurium S. typhi I III S. arizonae E. coli outgroup mdh gapA icd HGT “Clouds” Surrounding E. Coli and S. enterica subspecies I ASSORTATIVE YES mdh B21 B20 B34 B64 B25 B50 B8 B3 B21 B64 B34 B25 B20 B8 B3 B50 mutS mutS YES Recombination INTRAGENIC INTERSPECIES RECOMBINATION mdh mutS I VI II IV IIIB IIIA V ASSORTATIVE NO SARC 3333 INTRAGENIC mutS YES E. coli Salmonella E. coli Salmonella INTRA-SUBSPECIES RECOMBINATION (among S. enterica subspecies I strains) SARB INTRASPECIES RECOMBINATION (among S. enterica subspecies) SARC Genetic Distance mutS mdh ASSORTATIVE NO INTRAGENIC NO HGT “Clouds” Surrounding E. Coli and S. enterica subspecies I Whole Genome DNA Microarrays Salmonella enterica serovar Typhimurium LT2 Genome 4,857 kb 4,596 ORFs Salmonella Microarrays Containing ~4,500 PCRAmplified Salmonella Typhimurium Genes Supplementing the Typhimurium Microarray with Unique Genes from Salmonella Typhi and Salmonella Enteritidis: A Non-Redundant Microarray Representing Related Bacteria 471 Typhi ORFs Genes Unique to Typhi and Enteritidis added to Array 284 Enteritidis ORFs • NonRedundant Salmonella Enteritidis DNA Microarray: • 5184 Unique Genes per Array Spotted in Triplicate • 15,552 Spots Total per Slide Gene Expression or Genomic Comparison Studies Food Defense Finding a use for a method Is not synonymous with Finding a method that is useful Tiling Microarrays Pyrosequencing Optical Mapping Sampling ~1% of the E.coli O157:H7 Genome at Random 5.5 Mb Genome - Sampled 1 kb per ~100 kb - Tiled 60 Loci onto Arrays Perna, N.T. et al. Nature 409, 529-533 (2001) Interrogating 12 Independent Strains in Parallel 1.5 cm ~14,000 Spots (oligos) ~4mm 2 cm High Density Oligonucleotide Tiling Arrays Provide a “High Resolution” Snapshot of the Genome Reference Genome Test Genome 29-mer Tiling Array Probes Mutation • Our Tiled Strategy Uses a 5 nt Probe Spacing • For a random sampling of ~1% of the genome, 1 kb of genome sequence was selected at 60 equally spaced regions around the EDL933 chromosome. Relative Probe Intensity vs. Genome Position Probes reporting a deletion in the test strain Probes reporting a SNP in the test strain Probes reporting identical sequence between strains AB6 AB1 508 AB5 506 Pyrosequencing – Sequencing by Light CGT Polymerase CGT CGT CGT Pooled Genomic DNA AlleleSpecific PCR i Sulfurylase CGT CGT CAT 90% C Luciferase CGT CGT 10% T CGT C T strain serotype 4889b 4889a 5210 i559 roi NG1 NG2 NG7 4777 5096 barcode EC1214 O157:H7 A G C G G del T G G G 0000010100 EC506 O157:H7 A G C G A A T C G G 0000100000 EC868 O157:H7 A G C G A A T C G G 0000100000 86-24 O157:H7 A G C G A A T C G G 0000100000 EC509 O157:H7 A G C G A A T C A G 0000100010 EC536 O157:H7 A G C G A del T G G G 0000110100 EC484 O157:H7 A G C G GA A T C G G 0000200000 EC869 O157:H7 A G C A A del G G G G 0001111100 AB3 O157:H- A G C A A del G G G G 0001111100 EC510 O157:H- A G C A A del - - G G 0001112200 EC554 O157:H7 A G T G G A T C G C 0010000001 EC559 O157:H7 A G T G G A T C G C 0010000001 EC866 O157:H7 A G T G G A T C G C 0010000001 EC1219 O157:H7 A G T G G A T C G C 0010000001 95-01A O157:H7 A G T G G A T C G C 0010000001 AB1 O157:H- A G T G AG A T C G C 0010200001 EC558 O157:H7 A G T G GA A T C G C 0010200001 EC505 O157:H7 A A C G A A T C A G 0100100010 EC512 O157:H7 A A C G A - T C A G 0100120010 DEC7A O157:H43 G G C G G del T C G G 1000010000 EC521 O26:H11 G G C A G del G G G G 1001011100 EC1216 N.D. G G C A A del - - G G 1001112200 EC884 N.D. G G C A AG del - - G G 1001212200 DEC5C O55:H7 G G C - AG del G - G G 1002211200 Phylogenetic mapping of the roi gene G189A391 + + 1219 + + AB1 + + 554 + + + + 559 + + 95-001 + + 866 + + O157 Strains - + + - 1214 - - 484 - - AB3 + + + + 86-24 - + 868 + + DEC5A (O55:H7) - - roi DEC5C (O55:H7) - - roi 1216 - - roi 884 - - DEC7A (O157:H43) + + 521 (O26:H11) + - 1223 - - A roi AB roi B roi A189C391 B roi 69 B roi B roi B roi roi 506 roi 509 roi A roi A189A391 A roi roi A189A391 A roi roi G189A391 A roi 54 A189C391 roi roi 11 99 27 558 roi roi 59 512 510 100 roi II + B roi 100 505 + 86 E. coli O157:H7 SCCM 7 genes 3232 bp stx I A roi C roi 869 I II III IV V - CLADISTIC BIOMARKERS “CLADE-BREAKING” SEQUENCE-BASED “BINNING” DELINEATION OF PATHOGEN POPULATIONS USING REAL SEQUENCE CHANGES Exclusion S Y N A P O M O R P H Y A U T A P O M O R P H Y STRAIN-SPECIFIC UNIQUE ATTRIBUTE DELINEATION OF INDIVIDUAL PATHOGENIC STRAINS USING REAL SEQUENCE CHANGES Attribution E. coli K12 vs. E. coli O157:H7 Islands or Archipelagos? Mother Nature is the Quintessential Terrorist—she has been manipulating genomes for eons. Man, on the other hand, has been at it for just a couple of decades. We should look, therefore, to the “docking sites” of recombination that Mother Nature has used—these sites will be those likely to be used in strain manipulations. Optical Mapping: A Single Molecule Technique for Generating Whole Genome Restriction Maps Genome Map Optical Mapping: Image Analysis Single DNA molecule on Optical Chip after digestion, staining Image analysis software measures size and order of restriction fragments Converts “optical” data into digital data - barcodes Overlapping single molecule maps are aligned to produce a map assembly covering an entire chromosome Multiple Coverage is Necessary for Accurate Map Assembly Optical mapping; s#114-115 Sakai 1276 EDL933 1225 502 533 507 536 AB1 869 1231 s#129 s#168-169 Optical mapping; Inversions EC536-EDL933-Sakai 1,912,000 2,305,000 EC536 Sakai 2,225,000 Optical mapping; 502_EDL933 inversion e#151 EDL#151-161 151 15,289 bp 152 29,558 Inverted 502_EDL933 e#357 EDL#356-7 356 27,972 bp 357 17,040 bp Optical mapping; Inversions Sakai vs EDL933 vs EC533 Sakai vs EDL933 vs inverted map of EC533 Optical Maps are Well Suited for Strain Identification and Strain Relatedness Studies “Don’t think to hunt two hares with one dog.” --Ben Franklin